Particle-based immunoassay using a pegylated analyte-specific binding agent

ABSTRACT

Disclosed is a method for measurement of an analyte in a microparticle-based analyte-specific binding assay, wherein the microparticles are coated with the first partner of a binding pair, the method involving mixing the coated microparticles, an analyte-specific binding agent conjugated to the second partner of the binding pair, and a sample suspected of containing or containing the analyte, wherein the second partner of the binding pair is bound to the analyte-specific binding agent via a linker having from 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte via the conjugated analyte-specific binding agent to the coated microparticles, separating the microparticles having the analyte bound via the binding pair and the analyte-specific binding agent from the mixture and measuring the analyte bound to the microparticles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2016/069563 filed Aug. 18, 2016, which claims priority to EP15181763.2 filed Aug. 20, 2015, the disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a method for measurement of an analytein a microparticle-based analyte-specific binding assay, wherein saidmicroparticles are coated with the first partner of a binding pair, themethod comprising mixing the coated microparticles, an analyte-specificbinding agent bound to the second partner of the binding pair, and asample suspected of comprising or comprising the analyte, wherein saidsecond partner of the binding pair is bound to said analyte-specificbinding agent via a linker comprising from 12 to 30 ethylene glycolunits (PEG 12 to 30), thereby binding the analyte via theanalyte-specific binding agent to the coated microparticles, separatingthe microparticles comprising the analyte bound via the binding pair andthe analyte-specific binding agent from the mixture and measuring theanalyte bound to the microparticles.

Numerous methods and systems have been developed for the detection andquantitation of analytes of interest in biochemical and biologicalsamples. Methods and systems which are capable of measuring traceamounts of microorganisms, pharmaceuticals, hormones, viruses,antibodies, nucleic acids and other proteins are of great value toresearchers and clinicians.

Many assay methods make use of an analyte-specific binding agent tocapture a specific target molecule of interest from a sample and allowfor determination of the target molecule.

A substantial body of art has been developed based upon bindingreactions, e.g., antigen-antibody reactions, nucleic acid hybridizationtechniques, and protein-ligand systems. The high degree of specificityin many biochemical and biological binding systems has led to many assaymethods and systems of value in research and diagnostics. Typically, theexistence of an analyte of interest is indicated by the presence orabsence of an observable “label” attached to one or more of theanalyte-specific binding agents.

Assays sensitivity is largely limited by non-specific binding phenomena.Thus the main difficulty is to conceive an assay technology that is verysensitive and that at the same time does not intrinsically suffer from ahigh background signal, e.g. caused by the sample fluid that is probed.Non-specific binding typically leads to an increased background signal,to an inaccurate detection and to a higher (worse) detection limit. Inparticular, nonspecific binding is even more challenging when complexbiological matrices such as human plasma or serum samples are used assample fluids.

In recent years, more accurate and sensitive assays have been developed,which are based on the use of (e.g. superparamagnetic) microparticles.Especially in such particle-based assays, important contributions tononspecific signals come from particle-particle interactions and/or fromparticle-surface interactions.

U.S. Pat. No. 5,212,063 discloses a process for the detection ofanalytes in body fluids containing free biotin by immunoassay which makeuse of biotin conjugates. The document mentions polymer microparticlesconsisting of a core and containing a polymer which has a plurality ofbinding sites for biotin and, as a covering, at least one layer ofprotein.

WO 2013/001447 describes a precoated microparticle having a coatingcomprising a shell structure, wherein said shell structure comprises afirst layer comprising one or more affinity molecules (i.e. ananalyte-specific binding agent) and further a second layer, which iscoupled to the first layer, and wherein said first and second layercomprises non-affine spacer molecules forming a mesh, wherein said oneor more affinity molecules are embedded within the coating structure andwherein said mesh generates a steric hindrance for non-specificmolecules. Using such especially treated/coated microparticles thebackground caused by non-specific binding could be reduced.

However, even the most advanced assays still exhibit significant levelsof non-specific binding impairing either the lower limit of detection(LOD) or the measuring range or both. Test developers often have tocompromise between assay sensitivity, measuring range and assayspecificity. At the same time the tests need to be rapid, sensitive,quantitative accurate and even cost-effective. Moreover the platform onwhich the test is to be performed needs to be easy to use and reliable.

There is always a desire to improve assays by increasing ratio of signalto background noise and, therefore, the sensitivity of the assay.Increasing the signal of an assay also has several instrumentaladvantages including: i) less sensitive (and less expensive) detectionsystems are required; ii) smaller amounts of valuable samples arerequired; iii) instrumentation may be miniaturized so as to allow forinstruments that are smaller and/or devices that run many assaysconcurrently in a small area.

However, especially in particle-based assays, important contributions tononspecific signals come from particle-particle interactions andparticle-surface interactions.

There is thus a need to design novel structures and methods for improvedparticle-based assay methods. There is a strong need to avoidnon-specific binding to and clustering of microparticles, which is alimiting factor in a huge number of detection assays.

It has now surprisingly been found that linker molecules comprisingbetween 12 and 30 polyethylene glycol units—bound on the one hand to onemember of a binding pair and on the other hand to an analyte-specificbinding agent—can be used with great advantage in a microparticle-basedbinding assay, wherein the microparticles are coated with the othermember of the binding pair.

SUMMARY OF THE INVENTION

Disclosed is a method for measurement of an analyte in amicroparticle-based analyte-specific binding assay, wherein saidmicroparticles are coated with the first partner of a binding pair, themethod comprising a) mixing the coated microparticles, ananalyte-specific binding agent bound to the second partner of thebinding pair, and a sample suspected of comprising or comprising theanalyte, wherein said second partner of the binding pair is bound tosaid analyte-specific binding agent via a linker comprising from 12 to30 ethylene glycol units (PEG 12 to 30), thereby binding the analyte viathe analyte-specific binding agent to the coated microparticles, b)separating the microparticles comprising the analyte bound via thebinding pair and the analyte-specific binding agent from the mixture andc) measuring the analyte bound to the microparticles.

Also disclosed is a kit comprising in separate containers or inseparated compartments of a single container unit at leastmicroparticles coated with the first member of a binding pair and ananalyte-specific binding agent bound to the second member of thisbinding pair, wherein said second member of said binding pair is boundto said analyte-specific binding agent via a linker comprising from 12to 30 ethylene glycol units (PEG 12 to 30).

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic illustration of bead aggregation caused by ananalyte-specific binding agent bound to several biotin moieties (Bi) viaa state-of-the-art linker and binding to streptavidin molecules (crosseson the beads) on different beads.

FIG. 2 Schematic illustration of no bead aggregation due to use of ananalyte-specific binding agent bound to several biotin moieties (Bi) viaa linker comprising between 12 and 30 ethylene glycol units and bindingto streptavidin molecules (crosses on the beads) on the same bead.

FIG. 3 Pattern of beads on the measuring (working) electrode of anautomated Cobas® e601 analyzer (Roche Diagnostics GmbH) as obtained inan experimental HCV core antigen assay using Biotin-DDS forbiotinylation: The two pictures in the upper row represent the beaddistribution in a negative or control sample. The two pictures in thelower row represent the bead distribution in an HCV-positive sample. Thetwo pictures on the left hand side represent the bead distribution withmono-biotinylated antibody. The two pictures on the right hand siderepresent the bead distribution with a conjugate preparation obtained byusing 3.5 equivalents of biotinylation reagent per antibody in thepreparation of the conjugate. As obvious from the right hand picturesthe bead distribution is very uneven/disturbed, with most beads on theleft part of the electrode.

FIG. 4 Pattern of beads on the measuring (working) electrode of anautomated Cobas® e601 analyzer (Roche Diagnostics GmbH) as obtained inan experimental HCV core antigen assay using Biotin-PEG24-NHS forbiotinylation: The two pictures in the upper row represent the beaddistribution in a negative or control sample. The two pictures in thelower row represent the bead distribution in an HCV-positive sample. Thetwo pictures on the left hand side represent the bead distribution withmono-biotinylated antibody. The two pictures on the right hand siderepresent the bead distribution with a conjugate preparation obtained byusing 5 equivalents of biotinylation reagent per antibody in thepreparation of the conjugate. Even at this high ratio of biotinylationreagent per antibody the beads show a homogeneous distribution on theelectrode.

DETAILED DISCLOSURE OF THE INVENTION

In one embodiment the present disclosure relates to a method formeasurement of an analyte in a microparticle-based analyte-specificbinding assay, wherein said microparticles are coated with the firstpartner of a binding pair, the method comprising a) mixing the coatedmicroparticles, an analyte-specific binding agent bound to the secondpartner of the binding pair, and a sample suspected of comprising orcomprising the analyte, wherein said second partner of the binding pairis bound to said analyte-specific binding agent via a linker comprisingfrom 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding theanalyte via the analyte-specific binding agent to the coatedmicroparticles, b) separating the microparticles comprising the analytebound via the binding pair and the analyte-specific binding agent fromthe mixture and c) measuring the analyte bound to the microparticles.

Particle-based analyte-specific binding assays are widely used in e.g.certain nephelometric assays, certain latex agglutination assays andmany sensitive sandwich type assays employing a broad variety oflabeling or detection techniques.

A “particle” as used herein means a small, localized object to which canbe ascribed a physical property such as volume, mass or average size.Microparticles may accordingly be of a symmetrical, globular,essentially globular or spherical shape, or be of an irregular,asymmetric shape or form. The size of a particle envisaged by thepresent invention may vary. In one embodiment used are of globularshape, e.g. microparticles with a diameter in the nanometer andmicrometer range. In one embodiment the microparticles used in a methodaccording to the present disclosure have a diameter of 50 nanometers to20 micrometers. In a further embodiment the microparticles have adiameter of between 100 nm and 10 μm. In one embodiment themicroparticles used in a method according to the present disclosure havea diameter of 200 nm to 5 μm or from 750 nm to 5 μm.

Microparticles as defined herein above may comprise or consist of anysuitable material known to the person skilled in the art, e.g. they maycomprise or consist of or essentially consist of inorganic or organicmaterial. Typically, they may comprise or consist of or essentiallyconsist of metal or an alloy of metals, or an organic material, orcomprise or consist of or essentially consist of carbohydrate elements.Examples of envisaged material for microparticles include agarose,polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals,alloys or composition materials. In one embodiment the microparticlesare magnetic or ferromagnetic metals, alloys or compositions. In furtherembodiments, the material may have specific properties and e.g. behydrophobic, or hydrophilic. Such microparticles typically are dispersedin aqueous solutions and retain a small negative surface charge keepingthe microparticles separated and avoiding non-specific clustering.

In one embodiment of the present invention, the microparticles areparamagnetic microparticles and the separation of such particles in themeasurement method according to the present disclosure is facilitated bymagnetic forces. Magnetic forces are applied to pull the paramagnetic ormagnetic particles out of the solution/suspension and to retain them asdesired while liquid of the solution/suspension can be removed and theparticles can e.g. be washed.

The microparticles used in a method according to the present inventionare coated with the first member of a specific binding pair.

A “binding pair” as used herein consists of two partners binding to eachother with high affinity, i.e. with one nanomolar affinity or better.Embodiments for binding pairs are for example the binding pairsconsisting of receptor and ligand, hapten and anti-hapten antibody, andbinding pairs based on naturally occurring high affinity binding pairs.

One example of a receptor-ligand binding pair is a pair consisting of asteroid hormone receptor and the corresponding steroid hormone.

One type of a binding pair which is suitable for the method according tothe present invention is a hapten and anti-hapten antibody binding pair.A hapten is an organic molecule with a molecular weight of 100 to 2000Dalton, preferably of 150 to 1000 Dalton. Such small molecule can berendered immunogenic by coupling it to a carrier molecule andanti-hapten antibodies can be generated according to standardprocedures. The hapten may be selected from the group comprisingsterols, bile acids, sexual hormones, corticoids, cardenolides,cardenolide-glycosides, bufadienolides, steroid-sapogenines and steroidalkaloids, cardenolides and cardenolide-glycosides. Representatives ofthese substance classes are digoxigenin, digitoxigenin, gitoxigenin,strophanthidin, digoxin, digitoxin, ditoxin, and strophanthin. Anothersuitable hapten is for example fluorescein.

Examples of binding pairs based on naturally occurring high affinitybinding pairs are biotin or biotin analogues such as aminobiotin,iminobiotin or desthiobiotin and avidin or streptavidin as well as theFimG and DsF binding pair. The biotin-(strept)avidin binding pair iswell-known in the art. The basic principles of the FimG-DsF binding pairare e.g. described in WO2012/028697.

In one embodiment binding pairs are selected from hapten and anti-haptenantibody, biotin or biotin analogues such as aminobiotin, iminobiotin ordesthiobiotin and avidin or streptavidin, FimG and DsF, and receptor andligand.

In one embodiment binding pairs are selected from hapten and anti-haptenantibody and biotin or biotin analogues such as aminobiotin, iminobiotinor desthiobiotin/avidin or streptavidin, FimG and DsF.

In one embodiment the binding pair is biotin (or biotin analogues suchas aminobiotin, iminobiotin or desthiobiotin) and avidin orstreptavidin.

In one embodiment the binding pair consists of biotin and streptavidin.

In one embodiment the binding pair according to the present inventionconsists of a first partner of such binding pair having a molecularweight of 10 kD or more an of a second pair of such binding pair havinga molecular weight of 1 kD or less. As indicated above, the firstpartner of a binding pair, in one embodiment having a molecular weightof 10 kD or more, is bound (coated) to the microparticles used in amethod according to the present disclosure.

In one embodiment in the microparticle-based method according to thepresent disclosure said first partner of the binding pair is selectedfrom avidin and/or streptavidin, and FimG, respectively, and whereinsaid second partner of the binding pair is selected from biotin orbiotin analogues such as aminobiotin, iminobiotin or desthiobiotin andDsF, respectively.

In one embodiment in the microparticle-based method according to thepresent disclosure said first partner of the binding pair is avidinand/or streptavidin and wherein said second partner of the binding pairis biotin.

The microparticles used in a method according to the present inventionare “coated” with the first partner of a binding pair. Such coating isperformed according to state of the art procedures.

The first partner of the binding pair can be bound to the surface of theparticle by adsorption, by covalent binding or a combination of bothmethods. The microparticles optionally can be further incubated, e.g.with proteins, like bovine serum albumin, to reduce non-specific bindingof other assay components. The skilled artisan is fully aware of themethods used for such optional blocking of non-specific binding. In linewith the terminology used in the art, such coated and blockedmicroparticles also are simply referred to as coated microparticles.

The molecules of the first partner of the binding pair are present onthe microparticle in close proximity providing for many nearby bindingsites for the second partner of this binding pair. For mostpractical/routine applications the first partner of the binding pair iscoated to the microparticles at saturation concentration, resulting inan optimal coating density. As the skilled artisan appreciates, thecoating density, if desired could be reduced by using sub-optimalconcentrations of the first partner of a binding pair. In case asub-optimal concentration of the first partner of a binding pair wouldbe used for coating the person skilled in the art would choose theaverage coating density to match the linker length used for binding theanalyte-specific binding agent to the second partner of the bindingpair. In general terms: The average distance between the molecules of afirst partner of a binding pair on a coated microparticle will be atmost twice the length of the linker used for binding theanalyte-specific binding agent to the second partner of the bindingpair. The distance hereby is from the center of one molecule to thecenter of another molecule. As an example: The average length of apolyethylene glycol unit is about 0.38 nm. Thus a linker with 12PEG-units has about 4.5 nm in length. In order to allow the molecules ofthe second partner of a binding pair to bind to the first partners ofsaid binding pair on the same microparticle the average distance betweenthe molecules of the first partner of the binding pair on the particlewould be 9 nm or less. In one embodiment the average distance of themolecules of the first partner of a binding pair is 9 nm or less. In oneembodiment the average distance of the molecules of the first partner ofa binding pair is 9 nm or 8 nm, respectively. In one embodimentmicroparticles are used which have been coated at saturationconcentration of/with the first partner of a binding pair.

An “analyte” or “analyte of interest” or “target molecule” can be anymolecule which can be bound by an analyte-specific binding agent. In oneembodiment, an analyte within the context of the present invention is anucleic acid (DNA or RNA) molecule, a peptide, a protein, a drugmolecule, a hormone or a vitamin. In one embodiment, an analyte withinthe context of the present invention is a peptide, a protein, a drugmolecule, a hormone or a vitamin.

Liquid samples can be used in a method for specific in vitro-detectionof an analyte in a method according to the present disclosure. Thesample may be known to comprise the analyte or it may be suspected ofcomprising the analyte. In one embodiment a sample for in vitrodiagnosis used in a method according to the present disclosure is a bodyfluid selected from whole blood, blood serum, blood plasma, liquor,urine or saliva. In one embodiment the sample suspected of comprising orcomprising the analyte is serum, plasma or liquor. In one embodiment thesample suspected of comprising or comprising the analyte is serum orplasma.

The method for measurement of an analyte according to the presentdisclosure makes use of an analyte-specific binding agent. The term“analyte-specific binding agent” refers to a molecule specificallybinding to the analyte of interest. An analyte-specific binding agent inthe sense of the present disclosure typically comprises binding orcapture molecules capable of binding to an analyte (other terms analyteof interest; target molecule). In one embodiment the analyte-specificbinding agent has at least an affinity of 10⁷ l/mol for itscorresponding target molecule, i.e. the analyte. The analyte-specificbinding agent in other embodiments has an affinity of 10⁸ l/mol or evenof 10⁹ l/mol for its target molecule. As the skilled artisan willappreciate the term specific is used to indicate that other biomoleculespresent in the sample do not significantly bind to with the bindingagent specific for the analyte. In some embodiments, the level ofbinding to a biomolecule other than the target molecule results in abinding affinity which is only 10%, more preferably only 5% of theaffinity of the target molecule or less. In one embodiment no bindingaffinity to other molecules than to the analyte is measurable. In oneembodiment the analyte-specific binding agent will fulfill both theabove minimum criteria for affinity as well as for specificity.

In one embodiment the analyte-specific binding agent is selected fromthe group consisting of aptamers, peptide aptamers, proteins,oligonucleotides, and molecular imprinted polymers.

An “aptamer” as used within the context of an analyte-specific bindingagent may be a short nucleic acid molecule, e.g. an RNA, DNA, PNA, CNA,HNA, LNA or ANA molecule or any other suitable nucleic acid format knownto the person skilled in the art, being capable of binding an analyte.

Peptide aptamers are aptamers which are able to specifically bind to (a)protein(s), polypeptide(s) or peptide(s) comprising a specific aminoacid sequence. Typically, a peptide aptamer has a peptide loop,comprising for example 10 to 20 amino acids. In the context of thepresent disclosure a peptide aptamer may in specific embodiments beattached at one or both ends to a scaffold structure. The scaffoldstructure may be any molecule, preferably a protein, e.g. a protein,which has good solubility properties. Suitable scaffold molecules wouldbe known to the person skilled in the art. Example of suitable scaffoldmolecules are based on bacterial protein thioredoxin-A, and FkpA- orSlyD-chaperones, respectively. The aptamer peptide loop may preferablybe inserted within a reducing active site of the scaffold molecule.Alternatively, staphylococcal protein A and domains thereof andderivatives of these domains, such as protein Z or lipocalins may beused as peptide aptamers.

Nucleic acid or peptide aptamers may be generated according to anysuitable method known to the person skilled in the art, e.g. via PCR ormolecular synthesis approaches or yeast two-hybrid approaches.

A “peptide” as used within the context of an analyte-specific bindingagent may comprise or alliteratively consist of a stretch of 2 to 49amino acids, amino acid derivatives or a mixture thereof. The peptidemay be linear, branched, circular or mixture thereof. A peptidicanalyte-specific binding agent may also be attached to a scaffoldstructure as defined herein above.

A “polypeptide” or “protein” as used within the context of ananalyte-specific binding agent may comprise or alternatively consist ofa stretch of more than about 50 amino acids, amino acid derivatives or amixture thereof. The protein may have a linear, branched, and circularform or be comprised of a mixture of these forms.

In one embodiment the analyte-specific binding agent is a polypeptide ofat least 50 amino acids. Though in theory there is no upper limit to thepolypeptide length of an analyte-specific binding agent in oneembodiment will have at most 10.000 amino acids.

An “oligonucleotide” as used within the context of an analyte-specificbinding agent may comprise or alternatively consist of a stretch of 10to 120 nucleotides, or of 12 to 60, or of 15 to 40 nucleotides.

An oligonucleotide analyte-specific binding agent may be an RNA moleculeor a DNA molecule, or a mixture of both.

The term “molecular imprinted polymer” as used herein refers to apolymer which was formed in the presence of a molecule that is extractedafterwards, leaving a complementary cavity (an imprint) behind.Typically, a molecular imprinted polymer shows a certain chemicalaffinity for the original molecule. A molecular imprinted polymer may becomposed of any suitable polymeric unit known to the person skilled inthe art. Techniques for their production include polymerizationtechniques such as bulk, precipitation, emulsion, suspension,dispersion, gelation, multi-step swelling polymerization andhierarchical imprinting methods.

An “antibody” as used in the context of an analyte-specific bindingagent refers to an immunoglobulin molecule and to an immunologicallyactive portion (fragment) of an immunoglobulin molecule, i. e.antibodies or antibody fragments that contain an antigen binding sitethat immunospecifically binds the analyte. The immunoglobulin moleculesused in a method according to the present invention can be of any type(e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgG1, IgG2,IgG3, lgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.Antibodies can be described or specified in terms of the epitope(s) orportion(s) of a polypeptide which they recognize or specifically bind.Specific epitopes and their interaction with antibodies would be knownto the person skilled in the art.

The term “analyte-specific binding” as used in the context of anantibody refers to the immunospecific binding of an antibody to anepitope on the analyte. The concept of analyte-specific binding of anantibody via its epitope on an analyte is fully clear to the personskilled in the art.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies, singlechain antibodies, multispecific antibodies (e.g. bispecific antibodies)formed from at least two different antibodies, and antibody fragments solong as they exhibit the desired biological activity. An antibody in thesense of the present disclosure may also be part of a larger fusionmolecule, formed by covalent or non-covalent association of the antibodywith one or more other proteins or peptides.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purifiedto greater than 95% by weight of antibody, and in some embodiments, togreater than 99% as determined by SDS-PAGE under reducing or nonreducingconditions using, for example, Coomassie blue or silver stain.

Antibodies of the immunoglobulin G class usually are heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies among the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light-chain and heavy-chain variable domains.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

The antibodies used in a method according to the present invention maybe from any animal origin. In one embodiment the antibodies are human,murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig,camel, horse, or chicken antibodies.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of human immunoglobulins: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG2, IgG3, IgG4, IgA₁, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W. B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;single-chain antibody molecules; scFv, sc(Fv)2; diabodies; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody-hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies (sc(Fv)2). It is in this configuration that the three HVRs ofeach variable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHolliger et al., PNAS USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target-bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal-antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal-antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2^(nd) ed.1988); Haemmerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see,e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J.Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, PNAS USA 101(34): 12467-12472 (2004); and Lee et al., J.Immunol. Methods 284(1-2): 119-132(2004), and technologies for producinghuman or human-like antibodies in animals that have parts or all of thehuman immunoglobulin loci or genes encoding human immunoglobulinsequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO1991/10741; Jakobovits et al., PNAS USA 90: 2551 (1993); Jakobovits etal., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (e.g., U.S. Pat. No. 4,816,567 and Morrisonet al., PNAS USA 81:6851-6855 (1984)). Chimeric antibodies includePRIMATIZED® antibodies wherein the antigen-binding region of theantibody is derived from an antibody produced by, e.g., immunizingmacaque monkeys with the antigen of interest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all, or substantially all,of the FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino-acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., PNAS USA,103:3557-3562 (2006) regarding human antibodies generated via a humanB-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are Kabat complementarity-determining regions (CDRs) are basedon sequence variability and are the most commonly used (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)). Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat CDRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody-modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. Thevariable-domain residues are numbered according to Kabat et al., supra,for each of these extended-HVR definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

As disclosed herein, the second partner of the binding pair is bound tothe analyte-specific binding agent via a linker comprising from 12 to 30ethylene glycol units (PEG 12 to 30).

The term “linker” denotes a bifunctional or multifunctional moiety whichcan be used to conjugate (link) a first moiety with a second moiety ormore moieties. Conjugates comprising a first and a second moiety boundto each other can be conveniently prepared using a linker having tworeactive functionalities. In such conjugate the two moieties are bound“via” this linker. As obvious to the skilled artisan in such conjugatethe functional moieties of the linker are present as part of a bond andnot as an unreacted functional moiety.

Without wanting to be bound to the theory, it is believed that thelinker comprising from 12 to 30 ethylene glycol units is key to thesurprising findings disclosed herein. In the prior art onmicroparticle-based assays for measurement of an analyte only shortPEG-containing linker molecules are seriously considered. U.S. Pat. No.5,521,319 discloses a novel reagent that proved very useful forbiotinylation of biomolecules. The length of the linker is taught to beshort, i.e. up to 5 units of ethylene oxide, preferably of only 1 to 3units of ethylene oxide and most preferred of two such units. Contraryto this teaching it has now surprisingly been found that in amicroparticle-based analyte-specific binding assay long linkermolecules—comprising between 12 to 30 ethylene oxide units (=PEG 12 to30)—are advantageous if used to couple a the second member of a bindingpair, e.g. a biotin, to the analyte-specific agent.

An appropriate reagent for linking or coupling biotin via a PEG-linkerto a target molecule is for example a reagent according to Formula I

As will be appreciated (n) in Formula I relates to the number ofethylene glycol units. n preferably is from 12 to 30.

The methods of the invention may be constructed in a wide variety offormats. Such formats include formats known in the art such as sandwichassays and competitive binding assays (see, e.g., the followingreferences: Nonradioactive Labeling and Detection of Molecules, Kessler,C., ed., Springer-Verlag: Berlin 1992; The Immunoassay Handbook, Wild,D., ed., Stackton Press: New York 1994; Keller, G. H. and Manak, M. M.DNA Probes, 2nd Ed., MacMillan Publishers Ltd.: London, 1993; TietzTextbook of Clinical Chemistry 2nd Edition, Burtis et al. Ed., W.B.Saunders and Co.: Philadelphia, 1994).

In a method according to the present disclosure an analyte is measured.As the person skilled in the art will readily appreciate the measuringof the analyte bound to the microparticles is usually made bymeasurement of a signal carried or generated by a label on anappropriate assay component and by calculating the concentration of theanalyte from a standard curve for the analyte, i.e. thereby measuringthe analyte. The assay component to which a label is usually attached iseither a second analyte-specific binding agent (sandwich-type assays) orthe assay makes use of the competition between a labeled analyte and theanalyte in the sample. Before the label is measured, the microparticlescomprising part the labeled assay component are separated from the partof the labeled assay component not bound to the microparticles.

In one embodiment the methods of the present disclosure is practiced ina sandwich assay format.

A typically sandwich assay format includes mixing a microparticle coatedwith the first partner of a binding pair, an analyte-specific bindingagent bound to the second partner of the binding pair, a samplesuspected of comprising or comprising the analyte, wherein said secondpartner of the binding pair is bound to said analyte-specific bindingagent via a linker comprising from 12 to 30 ethylene glycol units (PEG12 to 30), and a second analyte-specific binding agent which isdetectably labeled. As obvious to the skilled artisan these componentsare mixed and incubated for a period of time sufficient for binding thedetectably labeled analyte-specific binding agent via the analyte, theanalyte-specific binding agent (bound to) the second partner of thebinding pair and the first partner of the binding pair to themicroparticles. In one embodiment, a sandwich assay without washingstep, such mixing/incubation is performed in a single reaction vessel.The sequence of adding and mixing the four ingredients (coatedmicroparticles, sample, analyte-specific binding agent bound to thesecond partner of the binding pair, and detectably-labeledanalyte-specific binding agent, respectively) is not critical. In oneembodiment, a sandwich assay with a washing step, the adding and mixingof microparticles coated with the first member of a binding pair, sampleand analyte-specific binding agent bound to the second partner of thebinding pair is performed in a single reaction vessel. After this first(analyte-capturing) step the microparticles to which the analyte is nowbound are washed before adding the detectably-labeled analyte-specificbinding agent. The sequence of adding and mixing of the first threeingredients (coated microparticles, sample and analyte-specific bindingagent bound to the second partner of the binding pair, respectively) isnot critical.

In a sandwich-type assay format in one embodiment the analyte-specificbinding agent bound to the second partner of the binding pair, and thedetectably-labeled analyte-specific binding agent, respectively, eachbind to the analyte at different and non-overlapping epitopes.

In one embodiment the methods of the present disclosure is practiced ina competitive assay format.

A typically competitive assay format includes mixing a microparticlecoated with the first partner of a binding pair, an analyte-specificbinding agent bound to the second partner of the binding pair, a samplesuspected of comprising or comprising the analyte, wherein said secondpartner of the binding pair is bound to said analyte-specific bindingagent via a linker comprising from 12 to 30 ethylene glycol units (PEG12 to 30) and an amount of analyte that is detectably labeled. Asobvious to the skilled artisan these components are mixed and incubatedfor a period of time sufficient for binding the fraction of thedetectably labeled analyte that—after competition by the analyte in thesample—is able to bind to the microparticles via the analyte-specificbinding agent (bound to) the second partner of the binding pair and thefirst partner of the binding pair coated to the microparticles.

Methods for labeling of an analyte-specific binding agent or of ananalyte are well-known to the person skilled in the art and abundantlydescribed e.g. in Haugland (2003) Molecular Probes Handbook ofFluorescent Probes and Research Chemicals, Molecular Probes, Inc.;Brinkley (1992) Bioconjugate Chem. 3:2; Garman, (1997) Non-RadioactiveLabeling: A Practical Approach, Academic Press, London; Means (1990)Bioconjugate Chem. 1:2; Glazer et al Chemical Modification of Proteins.Laboratory Techniques in Biochemistry and Molecular Biology (T. S. Workand E. Work, Eds.) American Elsevier Publishing Co., New York; Lundblad,R. L. and Noyes, C. M. (1984) Chemical Reagents for ProteinModification, Vols. I and II, CRC Press, New York; Pfleiderer, G. (1985)“Chemical Modification of Proteins”, Modern Methods in ProteinChemistry, H. Tschesche, Ed., Walter DeGruyter, Berlin and New York; andWong (1991) Chemistry of Protein Conjugation and Cross-linking, CRCPress, Boca Raton, Fla.); DeLeon-Rodriguez et al, Chem. Eur. J. 10(2004) 1149-1155; Lewis et al, Bioconjugate Chem. 12 (2001) 320-324; Liet al, Bioconjugate Chem. 13 (2002) 110-115; Mier et al BioconjugateChem. 16 (2005) 240-237.

The term detectably labeled encompasses labels that can be directly orindirectly detected.

Indirectly detectably labeled refers, e.g. to labeling with a hapten andto the detection of such haptenylated compound by an anti-haptenantibody carrying a directly detectable label or to the labeling with anenzyme and to the detection of such enzyme by its correspondingenzymatic activity resulting in the conversion of an appropriate dyesubstrate. Various enzyme-substrate labels are available or disclosed(see e.g. U.S. Pat. No. 4,275,149). The enzyme generally catalyzes achemical alteration of a chromogenic substrate that can be measuredusing various techniques. For example, the enzyme may catalyze a colorchange in a substrate, which can be measured spectrophotometrically.Alternatively, the enzyme may alter the fluorescence orchemiluminescence of the substrate. The chemiluminescent substratebecomes electronically excited by a chemical reaction and may then emitlight which can be measured (using a chemiluminometer, for example) ordonates energy to a fluorescent acceptor. Examples of enzymatic labelsinclude luciferases (e.g., firefly luciferase and bacterial luciferase;U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,malate dehydrogenase, urease, peroxidase such as horseradish peroxidase(HRP), alkaline phosphatase (AP), (3-galactosidase, glucoamylase,lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase,and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to polypeptides are describedin O'Sullivan et al “Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed. byJ. Langone & IT Van Vunakis), Academic Press, New York, 73 (1981)147-166.

Examples of enzyme-substrate combinations (U.S. Pat. No. 4,275,149; U.S.Pat. No. 4,318,980) include, for example: Horseradish peroxidase (HRP)with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidaseoxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or3,3′,5,5′-tetramethylbenzidine hydrochloride (TMB)); alkalinephosphatase (AP) with para-nitrophenyl phosphate as chromogenicsubstrate; and (3-D-galactosidase ((3-D-Gal) with a chromogenicsubstrate (e.g., p-nitro phenyl-(3-D-galactosidase) or fluorogenicsubstrate 4-methylumbelliferyl-(3-D-galactosidase.

Directly detectable labels either provide a detectable signal or theyinteract with a second label to modify the detectable signal provided bythe first or second label, e.g. to give FRET (fluorescence resonanceenergy transfer). Labels such as fluorescent dyes and luminescent(including chemiluminescent and electrochemiluminescent) dyes (Briggs etal “Synthesis of Functionalised Fluorescent Dyes and Their Coupling toAmines and Amino Acids,” J. Chem. Soc., Perkin-Trans. 1 (1997)1051-1058) provide a detectable signal and are generally applicable forlabeling. In one embodiment detectably labeled refers to a labelproviding or inducible to provide a detectable signal, i.e. to afluorescent label, to a chemiluminescent label or to anelectrochemiluminescent label, respectively.

In one embodiment according to the present disclosure themicroparticle-based analyte-specific binding assay makes use of achemiluminescent or an electrochemiluminescent label and a correspondinglight detection system. The light produced by the label is measured anddirectly or indirectly indicates the presence or quantity of theanalyte.

Electrochemiluminescent (ECL) assays provide a sensitive and precisemeasurement of the presence and concentration of an analyte of interest.Such techniques use labels or other reactants that can be induced toluminesce when electrochemically oxidized or reduced in an appropriatechemical environment. Such electrochemiluminescense is triggered by avoltage imposed on a working electrode at a particular time and in aparticular manner. The light produced by the label is measured andindicates the presence or quantity of the analyte. For a fullerdescription of such ECL techniques, reference is made to U.S. Pat. No.5,221,605, U.S. Pat. No. 5,591,581, U.S. Pat. No. 5,597,910, PCTpublished application WO90/05296, PCT published application WO92/14139,PCT published application WO90/05301, PCT published applicationWO96/24690, PCT published application US95/03190, PCT applicationUS97/16942, PCT published application US96/06763, PCT publishedapplication WO95/08644, PCT published application WO96/06946, PCTpublished application WO96/33411, PCT published application WO87/06706,PCT published application WO96/39534, PCT published applicationWO96/41175, PCT published application WO96/40978, PCT/US97/03653 andU.S. patent application Ser. No. 08/437,348 (U.S. Pat. No. 5,679,519).Reference is also made to a 1994 review of the analytical applicationsof ECL by Knight, et al. (Analyst, 1994, 119: 879-890) and thereferences cited therein. In one embodiment the method according to thepresent description is practiced using an electrochemiluminescent label.

As mentioned above electrochemiluminescense is triggered by a voltageimposed on a working electrode at a particular time and in a particularmanner. What is not mentioned in detail in the prior art is the fact thedistribution of microparticles on the working electrode has a majorimpact on the quality of an assay. The more aggregates in betweenmicroparticles are present—as a rule of thumb—the lower the quality ofone or more assay features. Aggregated particles often lead to a higherco-efficient of variation in between measurements, higher backgroundsignals and/or reduced assay sensitivity.

As can be easily imagined, the use of an analyte-specific binding agentbound to the second partner of the binding pair and comprising two ormore molecules of the second binding partner per molecule of theanalyte-specific binding agent can easily lead to aggregation ofmicroparticles coated with (many molecules of) the first partner of thebinding pair. Therefore in the prior art many attempts have been made toproduce conjugates consisting of one molecule of analyte-specificbinding agent and one molecule of the second partner of the bindingpair. Methods appropriate for obtaining such 1:1 conjugates are e.g.described in U.S. Pat. No. 6,391,571.

One of the most important recent approaches for site-specific proteinlabeling, especially site-specific mono-labeling of proteins is toincorporate bioorthogonal functionalities into these proteins atspecific sites via enzymatic reactions. For a recent review on“enzymatic labeling of proteins” see M. Rashidian et al., BioconjugateChemistry 24 (2013) 1277-1294. The enzymes used for site-specificconjugation covered in this review include formylglycine generatingenzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAcpost-translational modification, sortagging, transglutaminase,farnesyltransferase, biotin ligase, lipoic acid ligase, andN-myristoyltransferase.

Surprisingly, as shown throughout the examples section, ananalyte-specific binding agent bound to a single molecule of the secondpartner of the binding pair via a linker comprising from 12 to 30ethylene glycol units (PEG 12 to 30), e.g., a monobiotinylated antibody,leads to very good assay performance both regarding co-efficient ofvariation as well as signal-to noise ratio.

In addition the use of an analyte-specific binding agent bound to thesecond partner of the binding pair, wherein said second partner of thebinding pair is bound to said analyte-specific binding agent via alinker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30),appears not to require mono-biotinylation or removal of conjugates ofhigher than 1:1 stoichiometry obtained by use of standard couplingchemistry. As expected, conjugate preparations comprising conjugates ofhigher than 1:1 stoichiometry tend to lead to bead aggregation. However,this effect is much less visible, pronounced if the second partner ofthe binding pair is bound to said analyte-specific binding agent via alinker comprising from 12 to 30 ethylene glycol units (PEG 12 to 30),even at higher than 1:1 stoichiometry.

Without wanting to be bound to this theory a possible explanation mightbe that these relatively long and flexible PEG linkers allow for therapid binding of many of the second partners of the binding pair to thebinding sites of the first partner of such binding pair present andwithin reach on the coated microparticles. To the contrary, the severalsecond partners of a binding pair on an analyte-specific binding agentmay have too short linkers to bind to another first binding partner onthe same microparticle and rather tend to find an appropriate firstbinding partner on a second microparticle—thereby obviously promoting atendency for bead aggregation. FIGS. 1 and 2 are attempts toschematically illustrate this theory.

In order to avoid “over-labelling” with the second partner of thebinding pair the up to now standard chemistry must use a relatively lowratio of the analyte-specific binding agent and the second member of thebinding pair. In order to achieve a 1:1 stoichiometry of a conjugatepreparation in average usually the second partner of the binding pair(e.g. biotin in a biotinylation reagent) is used in 1.3-fold excess overthe analyte-specific binding agent (e.g. an antibody). At such 1.3 to 1coupling conditions the resulting conjugate preparation comprises about37% of non-conjugated antibody; about 37% of mono-biotinylated antibody,but also 18%, 6% and 2% of double-, triple- or more thantriple-biotinylated antibody, respectively. Usually the fractionrepresenting the 1:1-conjugate has to be purified for achieving optimalresults in a commercial immuno assay.

To the contrary, if the second partner of the binding pair is bound tosaid analyte-specific binding agent via a linker comprising from 12 to30 ethylene glycol units (PEG 12 to 30), standard coupling chemistry canbe used—even with the to be coupled/bound second member of the bindingpair at higher molar ratios—and not requiring the isolation of thefraction comprising the 1:1 conjugates. As obvious this, in addition tothe performance of such conjugates in the methods disclosed herein, is atremendous advantage in the production of such conjugates.

In one embodiment the present disclosure relates to a conjugatedspecific binding agent bound to the second partner of the binding paircomprised in a composition wherein in said composition the average molarratio between the second partner of the binding pair bound toanalyte-specific binding agent is 1.1 or more.

In one embodiment the present disclosure relates to a conjugatedspecific binding agent bound to the second partner of the binding paircomprised in a composition wherein in said composition the average molarratio between the second partner of the binding pair bound toanalyte-specific binding agent is between 1.1 and 10.

In one embodiment the present disclosure relates to a conjugatedspecific binding agent bound to the second partner of the binding paircomprised in a composition wherein in said composition the average molarratio between the second partner of the binding pair bound toanalyte-specific binding agent is between 1.2 and 6.

In one embodiment the present disclosure relates to a method formeasurement of an analyte in a microparticle-based analyte-specificbinding assay, wherein said microparticles are coated with the firstpartner of a binding pair, the method comprising a) mixing the coatedmicroparticles, an analyte-specific binding agent bound to the secondpartner of the binding pair, and a sample suspected of comprising orcomprising the analyte, wherein said second partner of the binding pairis bound to said analyte-specific binding agent via a linker comprisingfrom 12 to 30 ethylene glycol units (PEG 12 to 30), thereby binding theanalyte via the analyte-specific binding agent to the coatedmicroparticles and wherein said conjugated specific binding agent boundto the second partner of the binding pair is comprised in a compositionwherein in said composition the average molar ratio between the secondpartner of the binding pair bound to analyte-specific binding agent is1.1 or more b) separating the microparticles comprising the analytebound via the binding pair and the analyte-specific binding agent fromthe mixture and c) measuring the analyte bound to the microparticles.

In one embodiment the present invention relates to a kit comprising inseparate containers or in separated compartments of a single containerunit at least microparticles coated with a first partner of a bindingpair and an analyte-specific binding agent bound to the second partnerof said binding pair, wherein said second partner of the binding pair isbound to said analyte-specific binding agent via a linker comprisingfrom 12 to 30 ethylene glycol units (PEG 12 to 30).

The term single container unit relates to the fact that for manyautomatic analyzers, like the Elecsys® analyzer series from Rochediagnostics, the reagents required to measure a certain analyte areprovide in the form of a “reagent pack”, i.e. as one container unitfitting on the analyzer and containing in different compartments all thekey reagents required for measurement of the analyte of interest.

In one embodiment the present invention relates to a kit wherein saidfirst partner of a binding pair is avidin or streptavidin, and whereinsaid second partner of said binding pair is selected from biotin orbiotin analogues such as aminobiotin, iminobiotin or desthiobiotin.

In one embodiment the present disclosure relates to a kit comprising inseparate containers or in separated compartments of a single containerunit at least microparticles coated with avidin or streptavidin, and abiotinylated analyte-specific binding agent, wherein said biotin isbound to said analyte-specific binding agent via a linker comprisingfrom 12 to 30 ethylene glycol units (PEG 12 to 30).

The following examples, figures and sequences are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

Examples

Monoclonal antibodies are prepared by standard hybridoma technology asdescribed herein above or by recombinant NA techniques.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany). The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany).

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. full length antibodyheavy chain, full length antibody light chain, or an Fc-chain containingan oligoglycine at its N-terminus) a transcription unit comprising thefollowing functional elements is used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to beexpressed the basic/standard mammalian expression plasmid contains

-   -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.        Protein Determination

The protein concentration of purified polypeptides was determined bydetermining the optical density (OD) at 280 nm, using the molarextinction coefficient calculated on the basis of the amino acidsequence of the polypeptide or using the colorimetric BCA method.

Example 1

Synthesis of Activated Biotinylation Reagents

The synthesis of state-of-the-art activated biotin-comprising linkers(biotinylation reagents) like the widely used linker Biotin-DDS isdisclosed in EP 632 810.

Biotin-PEGn-NHS-biotinylation reagents (CAS-Nr. 365441-71-0; n=number ofethylene oxide units) were either obtained from IRIS Biotech GmbH orsynthesized in house. Biotin-PEG-110-NHS (Biotin-PEG-NHS, MW 5000 Da)was purchased from Iris Biotech GmbH (Germany).

In the de novo synthesis the control of discrete number of ethyleneoxide units was ensured by the stepwise elongation of shorter PEGs, suchas tetraethylene glycol, following the described method from Chen andBaker, J. Org. Chem. 1999, 64, 6840-6873.

In a first step bis-trityl-PEG_(n) 1 has been obtained (as obvious, nrepresents the number of ethylene glycol units).

Deprotection of 1 was carried out by stirring in 1M HCl in dioxane for 1h at room temperature. After evaporation the residue was refluxed inmethanol until a clear solution was obtained and the flask was kept at4° C. overnight. After filtration the solution was extracted withhexane, the methanolic layer evaporated and dried to give thecorresponding PEG_(n)-diol 2 as oil or wax (consistency depending onlength/number of units (n) of the PEG).

Next the introduction of the acid function was carried out by sodiumcatalyzed addition of PEG_(n)-diol to tert-butyl acrylate according toSeitz and Kunz, J. Org. Chem. 1997, 62, 813-826. This way compound 3 isobtained.

To a solution of HO-PEG_(n)-COOtBu 3 (1 equivalent) and triethylamine(2.5 equivalents) in methylene chloride methylsulfonyl chloride (2equivalents) was added drop-wise at 0° C. After stirring for 1 h aqueouswork-up and evaporation followed.

The mesylate 4 (1 equivalent) was directly reacted with NaN₃ (2equivalents) by stirring in dimethylformamide at room temperature fortwo days. After removal of the solids and dimethylformamide, aqueouswork-up with diethylether and Na₂CO₃ followed.

The crude product 5 was purified by column chromatography on silica inethyl acetate/methanol 15/1. Reduction of the azide 5 (1 equivalent) wasperformed by stirring for 24 h with triphenylphosphine (1.1 equivalents)in tetrahydrofurane/water 4/1 at room temperature. After evaporation theresidue was suspended in water and washed with ethyl acetate severaltimes. The water layer was evaporated and dried to give the amine 6 ascolorless oil.

Cleavage of the tert-butyl ester was carried out with 5% trifluoroaceticacid in water. Amino-PEG_(n)-acid 7 was obtained by evaporation withwater for several times.

Biotin was introduced by coupling with correspondingN-hydroxysuccinimide ester 8 (1.05 equivalents) with triethylamine (4equivalents) in dimethylformamide at room temperature overnight.

After evaporation crude product 9 was purified by RP-HPLC inacetonitrile/water.

Finally, N-hydroxysuccinimide ester 10 was formed by reaction withN-hydroxysuccinimide (1.1 equivalents) and ethyl dimethylaminopropylcarbodiimide (1.1 equivalents) in methylene chloride. After completionof the reaction, the reaction mixture was diluted with methylenechloride and washed with water. Evaporation and drying led to pureBiotin-PEG_(n)-NHS 10 as oil, wax or solid, respectively depending onnumber of ethylene oxide units.

Example 2

Coupling of Biotin and Ruthenium Moieties, Respectively, to Antibodies

Antibodies were obtained and purified according to state-of-theart-procedures that are fully familiar to a person skilled in the art.

For coupling, in general the lysine ε-amino groups of the antibodieswere targeted by N-hydroxy-succinimide activated compounds. At a proteinconcentrations of 10 mg/ml antibodies were reacted withN-hydroxy-succinimide activated biotinylation reagents andN-hydroxy-succinimide activated ruthenium labeling reagents,respectively. The molar ratio of biotinylation or labeling reagent,respectively/protein (antibody) varied from 1.3:1 to 5:1, depending onthe respective antibody conjugate. The reaction buffer was 50 mMpotassium phosphate (pH 8.5), 150 mM KCl. The reaction was carried outat room temperature for 30 minutes and stopped by adding L-lysine to afinal concentration of 10 mM. After the coupling reaction, unreactedfree biotin/label was removed by passing the crude antibody conjugatethrough a gel filtration column (Superdex 200 HI Load) or by dialysis.

In order to obtain mono-biotinylated antibody conjugates 0.5-1 Mammonium sulfate was added to the conjugate solution. The solution waspassed through a streptavidin mutein adsorber (see DE19637718)equilibrated with 50 mM potassium phosphate (pH 7.5), 150 mM KCl, 0.5-1M ammonium sulfate. Antibodies without any biotin coupled/bound to themare unable to bind the adsorber and were washed out. Mono-biotinylatedantibody conjugates were eluted with 50 mM potassium phosphate (pH 7.5),150 mM KCl and 2% DMSO. Antibody conjugates with more than one biotinper antibody were eluted with 50 mM potassium phosphate (pH 7.5), 150 mMKCl and 2 mM biotin (“+mSA”).

Example 3

Influence of the Linker in a Biotinylation Reagent on the ImmunologicalDetection of HCV Core Antigen

One and the same anti-HCV (capture) antibody was conjugated to biotin byuse of biotinylation reagents comprising various linkers. Theperformance of the different biotin-linker-antibody conjugates wasassessed on an automated Cobas® e601 analyzer (Roche Diagnostics GmbH).

Measurements were carried out in a sandwich assay format. Signaldetection in the Cobas® e601 analyzer is based onelectrochemiluminescense. In this sandwich assay the biotin-conjugate(i.e. the capture antibody) is immobilized on the surface of astreptavidin-coated magnetic bead (average bead size 2.8 μm). Thedetection-antibody bears a complexed ruthenium cation as the signalingmoiety. In the presence of analyte, the chromogenic ruthenium complex isbridged to the solid phase and emits light at 620 nm after excitation atthe platinum electrode comprised in the measuring cell of the Cobas®e601 analyzer. The signal output is in arbitrary light units.Measurements were performed with HCV core antigen positive and negativehuman serum and plasma samples purchased from several sources.

The experimental HCV core antigen assay was conducted as follows. 50 μlof normal or HCV antigen positive sample and 25 μl of a detergentcontaining pretreatment reagent to release the antigen were incubatedtogether for 9 minutes followed by the addition of 35 μl of 2 μg/mlcapture antibody-biotin conjugate and 40 μl of 1 μg/ml detectionantibody ruthenium label conjugate in the same physiological buffer ofpH 7.0 and comprising 100 mM potassium phosphate and 225 mM KCl. Afteradditional 9 minutes incubation time 50 μl streptavidin-coatedparamagnetic microparticles were added and incubated for further 9minutes. Afterwards, the HCV core antigen was detected (via theelectrochemiluminescent signal generated in these experiments).

TABLE 1 Comparison of different HCV capture antibody conjugates HCVcapture antibody conjugate Biotin- Biotin- DDS, Biotin- Biotin-PEG24,PEG24, mono DDS, 1:3.5 mono 1:5 counts counts counts counts normalsample 1 1,247 956 1,265 960 HCV antigen 742,023 1,162,932 1,792,0542,112,645 positive sample 1 S/N 595 1,216 1,417 2,201 normal sample 1(21-fold measurement) mean 1,302 949 1,298 973 min 1,243 894 1,233 933max 1,595 1,182 1,462 1,064 standard deviation 74 72 46 29 (SD)coefficient of 5.7% 7.6% 3.5% 3.0% variation (CV) HCV antigen positivesample 2 (21- fold measurement) mean 138,927 189,659 315,036 382,808 min125,384 177,906 308,373 373,402 max 143,586 199,343 320,929 392,804standard deviation 4,194 4,591 2,991 4,573 (SD) coefficient of 3.0% 2.4%1.0% 1.2% variation (CV)

Generally, a high signal to noise ratio (S/N) and a low coefficient ofvariation (CV) particularly for background signals are important assayperformance parameters especially regarding the lower limit of detectionfor an assay (assay sensitivity). As disclosed in EP 632 810 instreptavidin-coated microparticle based assays the Biotin-DDSbiotinylation reagent is preferred over other biotinylation reagentslike Biotin-NHS or Biotin-X-NHS comprising shorter or longer linkers,respectively.

In order to assess the bead distribution pattern on the workingelectrode the photomultiplier was replaced by a camera. In general beadaggregation of the paramagnetic streptavidin-coated microparticlesand/or an inhomogeneous bead distribution pattern during the magneticcapturing on the working electrode is known to lead to partial signalloss and especially to an increased CV. It is also known that so-calledmatrix effects present in some samples can further increase the problemscaused by bead aggregation.

The data in Table 1 show that an antibody conjugated with multipleBiotin-DDS moieties per antibody if compared to a conjugate of anantibody with only a single Biotin-DDS moiety has higher S/N values.However, this goes at the expense of a higher CV value for ananalyte-free serum as compared with its single biotinylated counterpart.Furthermore, multiple biotinylation of antibodies with Biotin-DDSinduces bead aggregation of the paramagnetic streptavidin-coatedmicroparticles becoming obvious by a very inhomogeneous beaddistribution pattern during the magnetic capturing on the workingelectrode (FIG. 3). (Since the negative serum used here is free ofmatrix effects, the negative impact on the CV is only moderate.) Thestrictly mono-biotinylated Biotin-DDS conjugate shows a much morehomogeneous bead pattern on the working electrode and hence it is lessprone to matrix effects (FIG. 3).

Using the Biotin-PEG24-NHS comprising a much longer linker forbiotinylation to produce a mono-biotinylated conjugate the signal tonoise ratio and the CV value could be increased and decreased,respectively. The bead pattern is very homogeneous, as for themono-conjugate based on Biotin-DDS (FIG. 4).

Surprisingly, with the Biotin-PEG24 mono-conjugate very good resultscould be obtained, both with respect to the signal-to-noise ratio aswell as regarding the co-efficient of variation. By using an antibodyconjugated with multiple Biotin-PEG24-NHS biotinylation reagents perantibody a homogeneous bead distribution pattern and low CV valuescomparable to the results obtained for the mono-biotinylated conjugatecould be obtained (FIG. 4). The signal to noise ratio rises with theincreasing number of biotin residues per antibody at least in the rangeof biotin residues per antibody used in these experiments.

Example 4

Influence of the Linker in a Biotinylation Reagent on the ImmunologicalDetection of Troponin T (TnT)

One and the same anti-troponin T (anti-TnT) (capture) antibody wasconjugated to biotin by use of biotinylation reagents comprising variouslinkers. The performance of the different biotin-linker-antibodyconjugates was assessed on an automated Cobas® e601 analyzer (RocheDiagnostics GmbH) based on an experimental protocol in line with theinstructions for the Elecsys Troponin T hs assay kit.

Measurements were carried out in a sandwich assay format. Signaldetection in the Cobas® e601 analyzer is based onelectrochemiluminescense. In this sandwich assay the biotin-conjugate(i.e. the capture antibody) is immobilized on the surface of astreptavidin-coated magnetic bead (average bead size 2.8 μm). Thedetection-antibody bears a complexed ruthenium cation as the signalingmoiety. In the presence of analyte, the chromogenic ruthenium complex isbridged to the solid phase and emits light at 620 nm after excitation atthe platinum electrode comprised in the measuring cell of the Cobas®e601 analyzer. The signal output is in arbitrary light units.

Measurements were performed with human serum supplemented with definedamounts of recombinant human cardiac TnT as calibrators and results aregiven in Table 2.

TABLE 2 Results obtained with an experimental troponinT-assay TnTcapture antibody conjugate TnT Biotin-DDS, Biotin-PEG24, Biotin-DDS,Biotin-PEG24, calibrators mono mono 1:1.3, +mSA 1:1.3, +mSA pg/ml countscounts counts counts 0 942 958 947 1,021 15 1,486 1,728 1,351 1,631 723,704 5,178 3,149 4,530 167 8,686 13,161 7,069 11,407 2,885 219,509414,720 201,995 380,924 10,910 840,288 1,566,614 793,111 1,576,584

It can be concluded from the data given in Table 2, that the captureantibody conjugates produced with the long Biotin-PEG24-NHSbiotinylation reagent are superior over the shorter Biotin-DDSbiotinylation reagent irrespective of the average ratio between biotinand antibody. (As explained in Example 1 a conjugate with the notion“+mSA” represents the fraction comprising two or more biotin moietiesper antibody.)

Example 5

Variation of Linker-Length in the Biotinylation Reagent on an HCV CoreAntigen Prototype Assay

In order to determine the optimal length of the linker differentBiotin-PEG(n)-NHS derivatives were conjugated with the HCV captureantibody and measured in the HCV core antigen experimental assay asdescribed in Example 3. For each biotinylation reagent bothmono-biotinylated and multiple biotinylated conjugates were generatedand results are given in Table 3.

TABLE 3 Influence of PEG-linker length on performance of an HCV coreantigen experimental assay normal HCV antigen positive HCV captureantibody sample 1 sample 3 conjugate counts counts S/N Biotin-PEG4, mono1,070 380,906 356 Biotin-PEG12, mono 970 550,556 568 Biotin-PEG24, mono884 605,566 685 Biotin-PEG30, mono 1,028 515,176 501 Biotin-PEG~110,mono 1,193 109,534 92 Biotin-PEG4, 1:1.3, +mSA 921 463,375 503Biotin-PEG12, 1:1.3, +mSA 956 707,700 740 Biotin-PEG24, 1:1.3, +mSA1,075 943,744 878 Biotin-PEG30, 1:1.3, +mSA 995 629,536 633Biotin-PEG~110, 1:1.3, 1,409 697,707 495 +mSA

As can be seen from Table 3, very good results as shown via thesignal-to-noise ratio (S/N) have been achieved with linkers comprisingbetween 12 and 30 ethylene glycol units. Variants generated by use ofthe Biotin-PEG24-NHS biotinylation reagent tended to show kind of anoptimal signal to noise ratio. However, as obvious from Table 3 linkerscomprising between 12 and 30 ethylene glycol units are quite superiorover the shorter as well as the longer linker also tested and yield goodresults.

The invention claimed is:
 1. A method for measurement of an analyte in amicroparticle-based analyte-specific binding assay, wherein saidmicroparticles are coated with the first partner of a binding pair, themethod comprising a) mixing the microparticles coated with the firstpartner of a binding pair with an analyte-specific binding agent boundto the second partner of the binding pair, and a sample suspected ofcomprising or comprising the analyte, wherein mixing of themicroparticles, analyte-specific binding agent and sample allows theanalyte-specific binding agent to bind to the microparticles coated withthe first partner of a binding pair, wherein said second partner of thebinding pair is bound to said analyte-specific binding agent via alinker comprising from 12 to 30 ethylene glycol units, thereby bindingthe analyte via the analyte-specific binding agent to the coatedmicroparticles, b) separating the microparticles comprising the analytebound via the binding pair and the analyte-specific binding agent fromthe mixture and c) measuring the analyte bound to the microparticles. 2.The method according to claim 1, wherein said microparticles are from 50nm to 20 μm in diameter.
 3. The method according to claim 1, whereinsaid microparticles are paramagnetic and separation in step (b) is bymagnetic forces.
 4. The method according to claim 1, wherein saidanalyte-specific binding agent is a polypeptide of at least 50 aminoacids.
 5. The method according to claim 1, wherein said analyte-specificbinding agent is a polypeptide of at most 10,000 amino acids.
 6. Themethod according to claim 1, wherein said analyte-specific binding agentis an antibody or an antigen-binding fragment thereof.
 7. The methodaccording to claim 1, wherein said analyte is measured in a competitiveassay format.
 8. The method according to claim 1, wherein said analyteis measured in a sandwich assay format.
 9. The method according to claim1, wherein said measuring of the analyte bound to the microparticles isbased on use of an electrochemiluminescent label.
 10. The methodaccording to claim 1, wherein said first partner of the binding pair isselected from avidin and/or streptavidin and protein FimG, and whereinsaid second partner of the binding pair is selected from biotin orbiotin analogues and protein DsF.
 11. The method according to claim 1,wherein said first partner of the binding pair is avidin and/orstreptavidin and wherein said second partner of the binding pair isbiotin.
 12. The method according to claim 1, wherein said specificbinding agent bound to the second partner of the binding pair iscomprised in a composition wherein in said composition the average molarratio between the second partner of the binding pair bound toanalyte-specific binding agent is 1.1 or more.